The living body of a mammal possesses humoral immunity which is a defense system for specifically capturing and eliminating various antigens (for example, exogenous antigens (viruses, bacterial toxins, and chemical substances), or autoantigens (for example, autoreactive lymphocytes; cancer cells; excessive endogenous factors (cytokines, hormones, or growth factors)) which are detrimental for maintaining homeostasis in the living body and can become causatives (pathogens) that cause or deteriorate various diseases. In this humoral immunity, the so-called antibodies (also called immunoglobulins) play a major role.
An antibody (immunoglobulin) has a Y-shaped basic structure comprising four polypeptide chains—two long polypeptide chains (immunoglobulin heavy chains; IgH chains) and two short polypeptide chains (immunoglobulin light chains; IgL chains). This Y-shaped structure is made when the two IgH chains bridged by disulfide bonds are connected to each of the IgL chains through another disulfide bond.
Due to this function of capturing and eliminating an antigen (pathogen) harmful to the living body, antibodies have been used as drugs for a long time. An early antibody drug was the so-called antiserum, and serum itself in which various types of antibodies against a specific antigen (for example, bacterial toxins and snake poison) are present (in other words, it was a polyclonal antibody) was used. The method for obtaining this antiserum however was limited to collecting from serum, and therefore, the supply was inevitably limited. Moreover, it was extremely difficult to isolate a single type of antibody molecule comprising specificity to a specific antigen, namely a monoclonal antibody, from this antiserum.
The successful preparation of a monoclonal antibody using hybridoma by Kohler and Milstein in 1975 (Nature, Vol. 256, p. 495-497, 1975) led to the solution of these problems.
Specifically, in this method, immortalized B cells (hybridoma) were obtained by fusing cells producing a monoclonal antibody of a specific type (B cells such as splenocytes) collected from a non-human mammal immunized by an antigen with myeloma cells, and thus immortalizing the cells. Then, the monoclonal antibody is purified after culturing the hybridoma. This method does not necessarily achieve mass-production of a desired monoclonal antibody, however, was a dramatic method as the desired monoclonal antibody can be obtained when it is desired. This technique enabled the use of monoclonal antibodies as drugs.
Monoclonal antibodies have been used as extremely useful drugs for preventing and treating various diseases due to their exceptional superiority in antigen specificity and stability in comparison to the above antiserums (polyclonal antibodies). They are also superior in their capability to control the biological activity of an antigen (for example, inhibition of activity, enhancement of activity, inhibition of signal transduction., signal transduction in place of ligands, or inhibition of intercellular adhesion) by specifically binding to the antigen (for example, exogenous antigens such as viruses and bacterial toxins; various endogenous factors such as cytokines, hormones, and growth factors, molecules on the cell surface such as receptors, cell adhesion molecule, and signal transduction molecules) involved in the onset or deterioration of diseases.
On the other hand, the amount of monoclonal antibody produced by the above hybridoma is not necessarily large, and it was difficult to produce a large amount of a monoclonal antibody by culturing the hybridoma and purifying and isolating the monoclonal antibody from the cell culture solution. Therefore, methods for producing a larger amount of monoclonal antibodies are being studied in order to cheaply supply a sufficient amount of monoclonal antibodies, which are extremely useful as drugs.
For example, it has been reported that antibody production increases when human antibody-producing hybridomas are cultured in an interleukin 2-supplemented culture medium (Cellular immunology, vol. 115, p. 325-333, 1988).
In addition, in an attempt using the genetic engineering technique, Ochi et al. have reported as follows (Proc. Natl. Acad. Sci. USA Vol. 80, p. 6351, 1983):
Recombinant cells obtained by introducing IgH chain gene (μ) and IgL chain gene (κ) encoding the IgH chain and IgL chain (κ), respectively, of an anti-hapten (TNP; 2,4,6-trinitrophenyl) monoclonal antibody, which were cloned from hybridoma Sp6 which produces mouse IgM monoclonal antibody specific to TNP into plasmacytoma X63Ag8 which produces a IgG monoclonal antibody against an unknown antigen different from the anti-TNP monoclonal antibody, produce both the anti-TNP antibody and the IgG antibody. Moreover, recombinant cells obtained by introducing IgH gene encoding the heavy chain of the anti-TNP antibody into mutant cells that derive from hybridoma Sp6 and secrete solely X chain, the light chain of the TNP antibody, but do not express the heavy chain and thus do not secrete the anti-TNP antibody as a result, produce the anti-TNP antibody.
However, the experiment of Ochi et al. failed to increase the amount of monoclonal antibody secreted, since the amount of anti-TNP-antibody produced by each of the above recombinant cells is about 10 to 25% of the amount of anti-TNP antibody produced by hybridoma Sp603 sub-cloned from the hybridoma Sp6.
In general, as a means to produce a large amount of a monoclonal antibody by hybridoma and host cells into which the antibody gene has been inserted, the method of increasing the number of cells per culture solution and the method of improving the production of a substance per cell have been studied.
The method of increasing the number of cells per culture solution is preferable, however, the increased number of cells does not necessarily lead to a high production of an antibody. It is important to select and isolate a single cell line with a high antibody productivity and culture the single cell line. Selection and isolation of cell lines with high antibody productivity (for example, hybridoma and recombinant cells) are laborious, but are extremely important factors for the purpose of increasing the productivity of cells producing a desired monoclonal antibody.
On the other hand, in the production of a desired protein using recombinant cells, the following method is used to increase the expression efficiency of a desired protein-encoding gene introduced into recombinant cells, and thus, increase the production of the desired protein. Namely, dihydrofolate reductase (DHFR) gene or glutamate synthase (GS) gene is introduced together with the gene encoding the desired protein into the recombinant cells to increase the copy number of the gene of the desired protein (for example, WO81/02426 and WO87/04462).
Taking the DHFR gene as an example, in these methods, specifically, an expression vector is constructed in which DHFR gene has been inserted near the gene encoding a desired protein, host cells are transformed with the expression vector, and drug-resistant lines are selected by culturing the host cells in the presence of a drug (for example, methotrexate (MTX), phosphinotricine, methionine sulfoximine).
In the obtained drug-resistant strains, the copy number of the introduced dhfr gene is increased (gene-amplified) and flanking genes are also amplified at the same time. It can be expected that, as a result of the amplified copy number of the gene encoding the desired protein, the desired protein production will also be increased.
In immortalized B cells (for example, hybridoma obtained by fusing the above B cells and myeloma cells) obtained by immortalizing monoclonal antibody-producing B cells isolated from a mammal immunized by an antigen, both the rearranged immunoglobulin heavy chain gene (IgH gene) and the rearranged immunoglobulin light chain gene (IgL gene) have been incorporated into the genome.
In order to amplify the IgH and IgL genes by the above gene-amplification gene, it is necessary to identify the location of each gene on the genome and to insert the gene-amplification gene near each gene.
This method is theoretically possible, however, it requires enormous time and labor and it is impossible to target the gene-amplification gene at a desired location on the genome of the immortalized B cells.